The long-term objective of this research is to elucidate the biochemical mechanisms underlying the circadian clock in Chlamydomonas reinhardtii. This green alga serves as a model system for a number of cell processes in animals and plants. Understanding its circadian clock will contribute to an understanding of circadian clock evolution and thus also help to better understand the circadian clock in humans. A precise knowledge of the circadian clock in humans is important for understanding health problems related to a defective clock like certain sleep disorders and depressions. The specific aim of this research is to identify the photoreceptor(s) responsible for adjusting the circadian clock to the daily light/dark cycles. The extent of adjustment will be monitored based on the circadian rhythm of phototaxis (swimming towards light) because its measurement has been automated. First experiments will determine the optimal time for giving a light pulse to yield the largest phase shifts in wild-type cells as well as the wavelength dependency at this optimal time. Previous analyses were performed with a cell-walless mutant and there is evidence that a wild-type strain shows major differences. To determine whether a particular photoreceptor is involved in entrainment, knockout strains with no photoreceptor protein or RNA interference strains with reduced photoreceptor levels will be tested for impaired abilities to phase shift upon light pulses. Two kinds of photoreceptors will be analyzed: rhodopsins and cryptochromes. Cryptochromes might also function as part of the central oscillator of the circadian clock. Cryptochrome-impaired strains might therefore exhibit rhythm impairments instead. If blue light entrains the circadian clock in wild-type cells, the single phototropin photoreceptor will also be tested. Rhodopsin knockout strains will be available from Peter Hegemann. RNA interference strains with reduced plant-like cryptochrome will be created by cloning available genomic and cDNA in reverse orientation and testing transformants with the available antiserum. The potential animal-like cryptochrome gene will be tested for expression by RT-PCR. Amplified cDNA will be cloned into an expression vector and the protein purified from E. coli to have antiserum made. Once all tools are available, RNA interference strains for animal-like cryptochrome can be created. RNA interference strains are already available for phototropin. Preliminary experiments indicate that they can be used in this study. The project will engage four undergraduates and two Master level graduate students stimulating their excitement for careers in the sciences, biomedical or otherwise. Understanding the circadian or biological clock in organisms like a green alga will help understand the clock in humans. This research should therefore also contribute to health-related questions like the role the clock plays in tumor suppression and how its defects can cause mental problems like some forms of depression and sleep disorders. It should also contribute to a better understanding of health problems due to shiftwork or jet lag and how to optimize work schedules or prepare for time zone travels in order to keep these problems to a minimum. [unreadable] [unreadable] [unreadable]